It is well known that most of the bodily states in mammals including most disease states, are affected by proteins. Such proteins, either acting directly or through their enzymatic functions, contribute in major proportion to many diseases in animals and man. Classical therapeutics has generally focused on interactions with such proteins in efforts to moderate their disease causing or disease potentiating functions. Recently, however, attempts have been made to moderate the actual production of such proteins by interactions with molecules that direct their synthesis, such as intracellular RNA. By interfering with the production of proteins, it has been hoped to affect therapeutic results with maximum effect and minimal side effects. It is the general object of such therapeutic approaches to interfere with or otherwise modulate gene expression leading to undesired protein formation.
One method for inhibiting specific gene expression is the use of oligonucleotides and oligonucleotide analogs as "antisense" agents. The oligonucleotides or oligonucleotide analogs complimentary to a specific, target, messenger RNA (mRNA) sequence are used. Antisense methodology is often directed to the complementary hybridization of relatively short oligonucleotides and oligonucleotide analogs to single-stranded mRNA or single-stranded DNA such that the normal, essential functions of these intracellular nucleic acids are disrupted. Hybridization is the sequence specific hydrogen bonding of oligonucleotides or oligonucleotide analogs to Watson-Crick base pairs of RNA or single-stranded DNA. Such base pairs are said to be complementary to one another.
Prior attempts at antisense therapy have provided oligonucleotides or oligonucleotide analogs that are designed to bind in a specific fashion to--which are specifically hybridizable with--a specific mRNA by hybridization. Such oligonucleotides and oligonucleotide analogs are intended to inhibit the activity of the selected mRNA--to interfere with translation reactions by which proteins coded by the mRNA are produced--by any of a number of mechanisms. The inhibition of the formation of the specific proteins that are coded for by the mRNA sequences interfered with have been hoped to lead to therapeutic benefits; however there are still problems to be solved. See generally, Cook, P. D. Anti-Cancer Drug Design 1991, 6,585; Cook, P. D. Medicinal Chemistry Strategies for Antisense Research, in Antisense Research & Applications, Crooke, et al., CRC Press, Inc.; Boca Raton, Fla., 1993; Uhlmann, et al., A. Chem. Rev. 1990, 90, 543.
Oligonucleotides and oligonucleotide analogs are now accepted as therapeutic agents holding great promise for therapeutics and diagnostics methods. But applications of oligonucleotides and oligonucleotide analogs as antisense agents for therapeutic purposes, diagnostic purposes, and research reagents often require that the oligonucleotides or oligonucleotide analogs be synthesized in large quantities, be transported across cell membranes or taken up by cells, appropriately hybridize to targeted RNA or DNA, and subsequently terminate or disrupt nucleic acid function. These critical functions depend on the initial stability of oligonucleotides and oligonucleotide analogs toward nuclease degradation.
A serious deficiency of unmodified oligonucleotides for these purposes, particularly antisense therapeutics, is the enzymatic degradation of the administered oligonucleotides by a variety of intracellular and extracellular ubiquitous nucleolytic enzymes.
A number of chemical modifications have been introduced into antisense agents--oligonucleotides and oligonucleotide analogs--to increase their therapeutic activity. Such modifications are designed to increase cell penetration of the antisense agents, to stabilize the antisense agents from nucleases and other enzymes that degrade or interfere with their structure or activity in the body, to enhance the antisense agents' binding to targeted RNA, to provide a mode of disruption (terminating event) once the antisense agents are sequence-specifically bound to targeted RNA, and to improve the antisense agents' pharmacokinetic and pharmacodynamic properties. It is unlikely that unmodified, "wild type," oligonucleotides will be useful therapeutic agents because they are rapidly degraded by nucleases.
Phosphorothioate modified oligonucleotides are capable of terminating RNA by activation of RNase H upon hybridization to RNA although hybridization arrest of RNA function may play some part in their activity. Phosphoramidites have been disclosed as set forth in U.S. patent application assigned to a common assignee hereof, entitled "Improved Process for Preparation of 2'-O-Alkylguanosines and Related Compounds," Ser. No. 918,362, the disclosures of which are incorporated herein by reference to disclose more fully such modifications. However, all reported modifications of the sugar-phosphate backbone, with the exception of phosphorothioates and phosphorodithioates, obliterate the RNase H terminating event. Cook, 1991, supra; Cook, 1993, supra; Uhlmann, et al., A. Chem. Rev. 1990, 90, 543. Heteroduplexes formed between RNA and oligodeoxynucleotides bearing 2'-sugar modifications, RNA mimics such as fluoro and alkoxys, do not support RNase H-mediated cleavage. These modified heteroduplexes assume an A form helical geometry as does RNA-RNA heteroduplexes which also do not support RNase H cleavage; Kawasaki, et al., J. Med. Chem., in press 1993; Lesnik, et al., Biochemistry, submitted 1993; Inoue, et al., Nucleic Acids Res. 1987, 15, 6131.
Oligonucleotides having phosphodiester linkages replaced by phosphorothioate residues have been demonstrated to be a sequence specific regulators of gene expression in eucaryotic and procaryotic systems. Chemical stability of these compounds in combination with efficient automatic synthesis, Iyer, et al., J. Am. Chem. Soc., 1990, 112, 1253-1254, results in the oligonucleotide phosphorothioates being the best to date candidates for practical application as "antisense" therapeutic agents. Eckstein, Oligonucleotide and Analogs, A Practical Approach, 1991, IRL Press, pp. 87-103.
Potential applications of phosphorothioate oligonucleotides as drugs have created a new challenge related to large-scale synthesis, purification, and analysis of the products. Although improvements in automatic assembling of phosphorothioate DNA analogs allows efficient synthesis of oligonucleotides, yet there are always occasional failures due to chemical and mechanical problems. Due to these problems, it is important to routinely confirm the sequence of these oligonucleotide products by direct sequence analysis. Replacing the non-bridging oxygen in the backbone with sulfur leads to desirable bio-chemical characteristics, including: resistance towards nucleases, retention of the ability to form double helix, solubility in water, chirality at phosphorus and stability to base catalyzed hydrolysis. Uhlmann, supra, 553-584. Unfortunately these features, advantageous in therapeutic technology, are disadvantageous from an analytical point of view, particularly problematic, is the chirality of the phosphorous atom.
In contrast to natural DNA, the phosphorus center of phosphorothioate oligonucleotides is chiral. Because of the stereospecificity of the coupling chemistry for oligonucleotide with many phosphorothioate linkages (n), there can be 2.sup.n possible diastereomers. Stereospecificity of nucleases for one or the other diastereoisomers of the phosphorothioate linkages varies. For instance S isomers are cleaved by nucleases P1 or S1, while R isomers can be cleaved by snake venom phosphodiesterase. Uhlmann, et al., Chem. Rev., 1990, 90, 4, 553-584. For the above reasons, direct cleavage of the oligonucleotide phosphorothioates with enzymes may lead to incomplete degradation, with yield influenced by variable, sequence specific populations of different stereoisomers. Described problems can be overcome by conversion of the oligonucleotide phosphorothioates into their phosphodiester analogs.
Several workers have studied replacing the sulfur atoms of the phosphorothioate oligonucleotides to attain the phosphodiesters. For example, it has been demonstrated that the sulfur in phosphorothioates can be replaced by oxygen when oxidation is carried out with iodine in pyridine in solution (phosphorothioate triesters are inert under these conditions). Connoly, et al., Biochem., 1984, 23(15), 3443-3453. There are also other methods available for desulfurization of phosphorothioates using 2-iodoethanol, Gish, Science, 1988, 240, 1520-1522; sodium metaperiodate, Agarval, et al., J. Chrom., 1990, 509, 396-399; iodine-bicarbonate, Burgers, et al., Biochem., 1979, 18, 450-454; cyanogen bromide, Sammons, et al., J. Biol. Chem., 1982, 257, 1138-1141; bromine, Lowe, et al., J. Chem. Soc., 1982, 595-598; and N-bromosuccinimide, Conoly, et al., J. Biol. Chem., 1982, 257, 3382-3384. Desulfurization of a phosphorodithioate dimer TpT was done using a solution of iodine in tetrhyrdofuran/water/n-methylimidazole. Porritt, et al., Tetrahedron Letters, 1990, 31(3), 1319-1322.
However, these methods were directed to oligonucleotides containing only one or two phosphorothioates; these methods are impractical because pharmaceutical formulations will typically contain many more nucleotide units. Desulfurized oligonucleotides have been typically analyzed using high performance liquid chromatography (HPLC) or enzymatic digestion; these methods are also impractical.
Oligonucleotides are typically characterized by sequencing to verify their composition. Generally, sequencing of short oligonucleotide phosphodiesters (10-25 size range) can be determined by partial enzymatic digestion ("wandering spot" method), Gait, Oligonucleotide Synthesis a Practical Approach, 1984, IRL Press, pp. 135-151, or by chemical sequencing, Maxam, et al., Gene, 1980, 10, 177. Chemical sequencing of oligonucleotides is based on the chemical modifications of the heterocyclic bases followed by cleavage of the oligonucleotide chain at the "modification" site under basic conditions.
Another limitation of the above described sequencing procedures, is that these procedures were designed to work in solution and need to be followed by time consuming and scale-limiting purification steps. This is also highly impractical.
DNA fragments have been sequenced on the ion-exchange cellulose-based carriers, Chuvpilo, et al., Bioorg. Khim., 1980, 9, 1694-1697, and C.sub.18 reverse-phase column, Jagadeeswaran, et al., Gene Anal. Tech., 1986, 3, 79-85. However, few attempts have been made to carry out sequencing on a solid support to eliminate losses and accelerate the process of sequence analysis.
Thus, there still remains a need for methods of characterizing oligonucleotides that are rapid, efficient, and readily adaptable to current pharmaceutical applications. The present invention addresses these, as well as other needs.